ABSTRACT Metastatic dissemination is the major cause of death in breast cancer patients. The majority of patients that develop metastatic disease present without clinically detectable metastases which, with the lack of reliable non-invasive methods for detection of early metastatic disease, remains the prevailing hurdle that prevents timely and effective therapeutic intervention in a well-defined patient population. One major player in invasion- metastasis cascade is a population of hematopoietic stem and progenitor cells (HSPC) that migrate from bone marrow to visceral organs where they organize and maintain pro-tumorigenic immunosuppressive microenvironment, known as pre-metastatic niche. HSPC is a heterogeneous population of continuously maturing cells, but at least one common marker has been identified as vascular endothelial growth factor receptor-1 (VEGFR-1). Pro-metastatic activity of HSPC can be blocked by a VEGFR1-specific antibody, indicating a critical role that VEGFR-1 plays in initiating metastatic disease. We hypothesize that targeted molecular imaging of VEGFR-1 could provide unique opportunities for detecting and characterizing early metastatic or even pre-metastatic events. Based on our previously published results involving the newly developed VEGFR-1 selective ligand scVR1, we envisage using the 18F- and 89Zr-labeled versions scVR1/F and scVR1/Zr, respectively, in PET imaging studies to gain new insight into the biology of metastatic dissemination. Our first goal within this study will address the evaluation of the target VEGFR-1 in terms of its prevalence and the mechanism involved in the VEGFR-1 mediated tracer uptake in both primary tumor as well as metastatic lesions. To that end, two metastasis models based on the murine breast cancer tumor cell line 4T1 will be utilized: spontaneous dissemination from primary orthotopic 4T1luc breast carcinoma and accelerated metastatic growth initiated by intracardiac injection of 4T1luc cells. VEGFR-1 prevalence will be determined from dose-dependent tracer uptake studies. Our receptor-selective tracer will then allow us to determine the receptor-specific uptake. Subsequent pretargeting experiments using in vivo click-chemistry will be used to distinguish between receptor-binding and receptor-mediated internalization mechanisms of tracer uptake. The second part of this study endeavors to answer the question, whether non-invasive imaging of VEGFR-1 allows for the detection of early metastasis. For that, we will utilize a specifically engineered 4T1luc/V model with enhanced expression levels of VEGF in combination with longitudinal PET imaging to label VEGFR-1-positive HSPC in vivo, at early stages of the invasion?metastasis cascade, prior to establishing of metastatic lesions. This new imaging approach would be instrumental in elucidating the role of VEGFR-1 positive cells in the onset and development of metastatic dissemination.